Use of (+)mefloquine for the treatment of malaria

ABSTRACT

Use of (+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol or a pharmaceutically acceptable salt thereof substantially free of its (−)-enantiomer in the manufacture of a medicament having reduced side-effect compared to the racemic (±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol for treating or preventing malaria in a subject. A method of treating or preventing malaria with reduced side-effects comprising administration of (+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol or a pharmaceutically acceptable salt thereof substantially free of its (−)-enantiomer.

The present invention relates to the use of an enantiomer ofα-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol(mefloquine) as an anti-malarial.

Malaria is an infectious parasitic disease transmitted by mosquitoes. Itis characterized by periodic fever and an enlarged spleen. Malariaaffects some 200 million people a year. Malaria in humans is caused by 4species of parasitic protozoa belonging to the genus Plasmodium. Ofthese, P. falciparum produces the severe disease while P. malariae, P.vivax and P. ovale cause milder forms.

Malaria is transmitted by infected female Anopheline mosquitoes. ThePlasmodia parasite matures in the insect, and is then transferred whenthe mosquito bites a human. Inside the human, the parasite settles firstin the liver, multiplies and then invades the red blood cells. This iswhen the symptoms of malaria become evident.

Despite numerous attempts at eradication, malaria remains a seriousendemic disease in many areas of Africa, Latin America and Oceania, witha worldwide mortality rate of approximately 1 million per year (WHOScientific Group on the Chemotherapy of Malaria 1990). One of the majorfactors contributing to the continued presence of malaria is theemergence of malaria parasites that are resistant to one or moreanti-malarial compounds.

Mefloquine is an anti-malarial compound which is effective againststrains of the Plasmodium parasite which have developed resistance toconventional anti-malarial agents (for a review of its antimalarialactivity, pharmacokinetic properties and therapeutic efficacy, seePalmer et al, Drugs, 1993, 45, 430-475). However, mefloquine resistancehas now been reported in a number of areas including areas of Thailand(see Palmer et al.). Nevertheless, mefloquine is still one of the mosteffective anti-malarial mono-therapies and its use has increasedgreatly. Recently, the drug has attracted considerable adverse publicityowing to the incidence of severe neuropsychiatric side-effects, e.g.depression, psychosis, panic attacks, generalised anxiety. Althoughcentral nervous system (CNS) side-effects had been reported previously(particularly from its use by the armed forces in tropical areas; Croft& World (Neuropsychiatric reactions with mefloquine chemoprophylaxis.Lancet, 1996, 347, 326); Gullahorn et al. (Anaesthesia emergencedelerium after mefloauine prophylaxis. Lancet, 1993, 341, 632)), theirincidence had been regarded as sufficiently low to be of little concern.However, the widespread use of the drug by holidaymakers has resulted ina greatly increased number of CNS side-effect reports. A recent study(Barrett et al. (Comparison of adverse events associated with use ofmefloquine and combination of chloroquine and proguanil as antimalarialprophylaxis: postal and telephone survey of travellers. (British MedicalJournal, 1996, 313, 525-528), in which 3851 travellers takingprophylactic anti-malarial medication were surveyed, has confirmed thatthere is a significant excess of adverse neuropsychiatric eventsassociated with mefloquine administration compared with an alternativeprophylactic treatment (proguanil plus chloroquine).

Clinical reports indicate that mefloquine may be proconvulsant (Ruff etal. (Seizure associated with mefloquine for malaria prophylaxis. Med. J.Aust., 1994, 161, 453)), anxiogenic (Hennequin et al. (Severepsychiatric side effects observed during prophylaxis and treatment withmefloquine. Arch. Intern. Med., 1994, 154, 2360-2362)), induce vertigoand dizziness (Sowunmi et al. (Neuropsychiatric side effects ofmefloquine in Africans. Trans. Roy. Soc. Trop. Med. Hyg., 1993, 87,462-463)) and may have central anticholinergic actions (Speich andHeller (Central anticholinergic syndrome with the antimalarial drugmefloquine. N. Engl. J. Med., 1994, 331, 57-58)).

Mefloquine is a molecule having two asymmetric carbon atoms and isusually used clinically as a racemic mixture of erythro ((±) (R*,S*))isomers. Both of the mefloquine enantiomers have been reported to beequally effective against Plasmodium falciparum (Basco et al. (In vitroactivity of the enantiomers of mefioquine, halofantrine and enpirolineagainst Plasmodium falciparum. Br. J. clin. Pharmac., 1992, 33,517-520)), although another study claimed that the (+)-enantiomer wasmore potent than the (−)-enantiomer by a factor of 1.69-1.81 (Karle etal. (Plasmodium falciparum: role of absolute stereochemistry in theantimalarial activity of synthetic amino alcohol antimalarial agents.Exp. Parasitol., 1993, 76, 345-351)).

Mefloquine hydrochloride was introduced to the market as ananti-malarial agent in 1985. There have been over 40 families of patentapplications on mefloquine covering: the use of the compound fortreating malaria and other parasitic diseases; various processes for itspreparation; and different formulations. The compound and itspreparation was first described by Ohnmacht et al. (J. Med. Chem. 1971,14, 926) in 1971. A more detailed account of the stereochemistry,synthesis, and anti-malarial activity of the isomers of mefloquine isgiven by Carroll and Blackwell (J. Med. Chem. 1974, 17, 210-219) in1974.

It has now been found that the (+)-(11R,2′S)-enantiomer ofα-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol iseffective as an anti-malarial and has reduced side-effects compared tothe racemic(±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.The structures of the (+)-(11R,2′S)-enantiomer and the(−)-(11S,2′R)-enantiomer ofα-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol are shownbelow.

In particular, it has been found that the (−)-enantiomer of mefloquinebinds to CNS adenosine receptors, while the (+)-enantiomer is withoutsignificant activity at this binding site. The blocking of centraladenosine receptors by the (−)-enantiomer is believed to result in theneuropsychiatric symptoms associated with mefloquine.

According to the present invention there is provided use of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolor a pharmaceutically acceptable salt thereof substantially free of its(−)-enantiomer in the manufacture of a medicament having reducedside-effects compared to the racemic(±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolfor treating or preventing malaria.

According to a further aspect of the present invention there is provideda method of treating or preventing malaria with reduced side-effectscompared to the racemic(±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolcomprising administration to a subject in need of such treatment aneffective dose of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolor a pharmaceutically acceptable salt thereof substantially free of its(−)-enantiomer.

The present invention may be employed in respect of a human or animalsubject, more preferably a mammal, more preferably a human subject.

(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolmay be employed in the present invention in an admixture with one ormore other anti-malarial drugs such as, for example, chloroquine,pyrimethamine, sulfadoxine, amodiaquine, quinine/quinidine,halofantrine, artemether/artesunate, tovaquone, proguanil, doxycyclineand dapsone. Combination with pyrimethamine and sulfadoxine isparticularly preferred. According to a further aspect of the invention,(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanoland the other anti-malarial drug(s) may be in separate formulations, foruse simultaneously or sequentially.

The term “a method for treating or preventing malaria” as used herein,means relief from malaria, preventing or inhibiting infection byparasitic protozoa of the genus Plasmodium which cause malaria andclearance of parasitic protozoa.

The term “substantially free of its (−)-enantiomer”, as used herein,means that the composition contains a greater proportion of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolin relation to(−)-(11S,2′R)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.In a preferred embodiment of the present invention the term“substantially free of its (−)-enantiomer” as used herein means that thecomposition contains at least 90% by weight of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanoland 10% by weight or less of(−)-(11S,2′R)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.In a further preferred embodiment, the term “substantially free of its(−)-enantiomer” means that the composition contains at least 99% byweight of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanoland 1% or less of(−)-(11S,2′R)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.In another preferred embodiment, the term “substantially free of its(−)-enantiomer” as used herein means that the composition contains 100%by weight of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.The above percentages are based on the total amount ofα-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol present inthe composition.

The term “reduced side-effects”, as used herein, means that thepharmaceutical compositions employed in the present invention allowtreatment and prevention of malaria and cause less side-effects than theracemic(±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.In a preferred embodiment of the present invention the term “reducedside effects”, as used herein, means that the pharmaceuticalcompositions employed in the present invention allow treatment andprevention of malaria and cause substantially less side-effects than theracemic(±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.In a more preferred embodiment of the present invention the term“reduced side-effects”, as used herein, means that the pharmaceuticalcomposition employed in the present invention allow treatment andprevention of malaria and cause substantially no side-effects. Theside-effects that are reduced, preferably substantially reduced, andmore preferably avoided, include neuropsychiatric side-effects such asdepression, psychosis, irritability, aggressiveness, panic attacks andgeneralised anxiety; seizures; proconvulsant effects; agitation effects;vertigo; dizziness; and anticholinergic symptoms. Preferably, thepharmaceutical compositions employed in the present invention exhibitreduced, preferably substantially reduced, preferably eliminated sideeffects associated with purinergic receptor binding activity.

It has been found that the (−)-enantiomer binds strongly to theadenosine receptor, while the (+)-enantiomer exhibits only a weakbinding interaction.

There is evidence that blockade of adenosine receptors can result in anumber of neuropsychiatric symptoms including tremor, anxiety, panic,insomnia and convulsions.

Adenosine receptors represent a sub-class (P₁) of the group of purinenucleotide and nucleoside receptors known as purinoreceptors. The mainpharmacologically distinct adenosine receptor subtypes are known as A₁,A_(2A), A_(2B) (of high and low affinity) and A₃. Recent studies on micebred without the adenosine A_(2A) receptor (Ledent et al.,(Aggressiveness, hypoalgesia and high blood pressure in mice lacking theadenosine A_(2A) receptor. Nature, 1997, 388, 674-678)) indicate thatthese animals are more aggressive and anxious as well as having higherblood pressure than normal mice.

The effects in humans and laboratory animals of the adenosine receptorantagonists of theophylline, an asthma drug, and caffeine are welldocumented. These two drugs have fairly weak in vitro affinities atadenosine receptors (Ki values: theophylline A₁ 8.5 μM, A_(2A), 25 μM;caffeine A₁ 29 μM, A_(2A), 48 μM) (Jacobson, K. A. and van Rhee, A. M.(Development of Selective Purinoceptor Agonists and Antagonists, inPurinergic Approaches in Experimental Therapeutics, K. A. Jacobson andM. Jarvis (Eds). John Wiley and Sons, Inc., New York, 1997., pp101-128)).

Patients taking theophylline are warned that they should inform theirphysicians if they have a seizure disorder, which is not surprising inview of the seizure inducing potential of adenosine receptorantagonists. Overdose with xanthines can result in seizures that areparticularly refractory to treatment with clinically usefulanticonvulsants (Chu, C. K. (Caffeine and aminophylline inducedseizures. Epilepsia 1981, 22, 85-94)).

Adenosine is thought to be the agent that causes seizure arrest in man(Knutsen, L. J. S. and Murray, T. Adenosine and ATP in Epilepsy. In“Purinergic Approaches in Experimental Therapeutics”, K. A. Jacobson andM. Jarvis (Eds)., John Wiley and Sons, Inc., New York, 1997, pp423-447), and experimental data from rodents indicates that blockade ofadenosine A₁ receptors by theophylline immediately prior to an ischaemicepisode exacerbates neuronal damage (Rudolphi, K. A.; Keil, M.; Hinze,H. J. Effect of theophylline on ischemically induced hippocampal damagein Mongolian Gerbils:, a behavioral and histopathological study. J.Cereb. Blood Flow Metab. 1987, 7, 74-81).

Given the undesired CNS effects observed in mammals with the relativelyweak adenosine receptor antagonists theophylline and caffeine documentedin the scientific literature, it is undesirable to administer to normalhuman subjects a potent adenosine receptor antagonist.

According to the present invention the use of the (+)-enantiomer ofmefloquine, substantially free of the (−)-enantiomer, is particularlypreferred in the treatment of a subject who has consumed or is likely toconsume any chemical compound which interacts with a purinergicreceptor. Consumption of such a compound includes ingestion of such acompound in food or drink, and administration of a drug, for example fortreatment or prophylaxis of a medical condition by any route ofadministration (such as, but not limited to, oral ingestion). Compoundswhich interact with purinergic receptors particularly include compoundswhich interact with adenosine receptors. Such compounds includecaffeine, theobromine, theophylline, carbamazepine, papaverine anddipyridamole; particularly caffeine, theobromine, theophylline,carbamazepine and papaverine; more particularly, caffeine, theobromine,theophylline and carbamazepine; more particularly caffeine, theobromineand theophylline; more particularly caffeine and theobromine; mostparticularly caffeine. Compounds which interact with purinergicreceptors also include ethanol.

According to a further aspect of the present invention, the use of the(+)-enantiomer of mefloquine, substantially free of the (−)-enantiomer,is particularly preferred in the treatment of a subject having, or atrisk from, a medical condition related to a disorder of purinergicreceptor function. Medical conditions related to purinergic receptoractivity include conditions causing or caused by an imbalance inpurinergic receptor activity and conditions for which the medicationcauses an imbalance in purinergic receptor activity. Such medicalconditions include, for example, neuropsychiatric disorders,cardiovascular disorders, digestive disorders, bronchial disorders anderectile dysfunction. Neuropsychiatric disorders of the CNS includepsychiatric disorders [such as depression and anxiety disorders (such aspanic disorders)], sleep disorders and seizure disorders. Cardiovasculardisorders, which may be diagnosed or undiagnosed, include ischaemicheart disease, arrhythmia and related disorders, congestive heartfailure and cerebrovascular disease (including transient ischaemicattacks). Subjects having cardiovascular disorders (or cardiovascularsystem investigations) particularly include subjects undergoingtreatment with a beta-blocker drug, vasodilator or nifedipine. Subjectshaving digestive disorders particularly include subjects undergoingtreatment with proton pump inhibitors. Bronchial disorders particularlyinclude asthma and chronic obstructive pulmonary disease. Subjectshaving bronchial disorders particularly include subjects undergoingtreatment with theophylline.

The pharmaceutical compositions employed in the present inventioncomprise(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolas an active ingredient or a pharmaceutically acceptable salt thereofand may also contain a pharmaceutically acceptable carrier andoptionally other therapeutic ingredients known to those skilled in theart, such as one or more other anti-malarial drugs such as, for example,chloroquine, pyrimethamine, sulfadoxine, amodiaquine, quinine/quinidine,halofantrine, artemether/artesunate, tovaquone, proguanil, doxycyclineand dapsone. The term, “pharmaceutically acceptable salts”, refers tosalts prepared from pharmaceutically acceptable non-toxic acidsincluding inorganic acids and organic acids.

Since the compound employed in the present invention is basic, salts maybe prepared from pharmaceutically acceptable non-toxic acids includinginorganic and organic acids. Such acids include acetic, benzenesulfonic,benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic,glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic andthe like. Particularly preferred are hydrochloric, hydrobromic,phosphoric, and sulfuric acids, and most particularly preferred is thehydrochloride salt.

Any suitable route of administration may be employed for providing thepatient with an effective dosage of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol.For example, oral, rectal, parenteral (intravenous, intramuscular),transdermal, subcutaneous, and the like may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,patches, and the like. The most suitable route in any given case willdepend on the severity of the condition being treated. The mostpreferred route of administration of the present invention is the oralroute. The compositions may be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

In practical use,(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolcan be combined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.oral or parenteral (e.g. intravenous). In preparing the compositions fororal dosage form, any of the usual pharmaceutical media may be employedas carriers, such as, for example, water, glycols, oils, alcohols,flavouring agents, preservatives, colouring agents, and the like in thecase of oral liquid preparations (such as suspensions, solutions andelixirs) or aerosols; or carriers such as starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like may be used in the case oforal solid preparations such as, for example, powders, capsules, andtablets, with the solid oral preparations being preferred over theliquid preparations. The most preferred solid oral preparation istablets.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are employed. If desired, tablets may be coatedby standard aqueous or non-aqueous techniques.

In addition to the common dosage forms set out above, the compounds ofthe present invention may also be administered by controlled releasemeans and/or delivery devices such as those described in U.S. Pat. Nos.:3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200; 4,008,719;4,687,660; and 4,769,027, the disclosures of which are herebyincorporated by reference.

Pharmaceutical compositions employed in the present invention suitablefor oral administration may be presented as discrete units such ascapsules, cachets, or tablets, or aerosol sprays each containing apredetermined amount of the active ingredient as a powder or granules, asolution or a suspension in an aqueous liquid, an oil-in-water emulsion,or a water-in-oil liquid emulsion. Such compositions may be prepared byany of the methods of pharmacy, but all methods include the step ofbringing the active ingredient into association with the carrier whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation.

For example, a tablet may be prepared by compression or moulding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with a binder, a lubricant, an inert diluent, and/or a surfaceactive or dispersing agent. Moulded tablets may be made by moulding in asuitable machine a mixture of the powdered compound moistened with aninert liquid diluent.

The invention is further defined by reference to the following Figuresand examples. It will be apparent to those skilled in the art that manymodifications, both to materials and methods, may be practised withoutdeparting from the purpose and interest of this invention.

FIG. 1 shows binding of the mefloquine (+)- and (−)-enantiomers tobovine striatal adenosine receptors (non-selective) (displacement of[³H]-NECA ([³H]-5′-N-ethylcarboxamidoadenosine)). Table 2 displayscomparative data for the control compound MECA(5′-N-methylcarboxamidoadenosine).

FIG. 2 shows binding of the mefloquine (+)- and (−)-enantiomers to ratbrain adenosine A₁ receptors (displacement of [³H]-DPCPX, an adenosineA₁ receptor antagonist).

FIG. 3 shows binding of the mefloquine (+)- and (−)-enantiomers to humanrecombinant (HEK-293) adenosine A_(2A) receptors (displacement of[³H]-CGS 21680, an adenosine A_(2A) receptor agonist).

FIG. 4 shows binding of the mefloquine (+)- and (−)-enantiomers to humanrecombinant (HEK-293) adenosine A₃ receptors (displacement of[¹²⁵]AB-MECA, an adenosine A₃ receptor agonist).

FIG. 5 shows the effect of the mefloquine (+)- and (−)-enantiomers onthe NECA-induced increase in extracellular acidification rate (ECAR) inPC-12 cells.

FIG. 6 shows the effect of the mefloquine (+)- and (−)-enantiomers onthe CGS 21680-induced increase in ECAR in PC-12 cells.

EXAMPLES Preparation of 2,8-bis(Trifluoromethyl)quinoline-4-carboxylicAcid

The compound 2,8-bis(trifluoromethyl)quinoline-4-carboxylic acid wasprepared by the method of Hickmann et al (U.S. Pat. No. 4,327,215).

Preparation ofN-Methoxy-N-methyl-2,8-bis(trifluoromethyl)-quinoline-4-carboxamide

This compound was prepared using synthetic methodology reported byThiesen et al (J. Org. Chem. 1988, 53, 2374). To a suspension of2,8-bis(trifluoromethyl)quinoline-4-carboxylic acid (12.5 g, 40.4 mmol)in CH₂Cl₂ (200 ml) was added 1,1′-carbonyldiimidazole (7.3 g, 45 mmol)and N,O-dimethylhydroxylamine hydrochloride (4.25 g, 45 mmol). Theresulting deep red solution was stirred overnight, then poured intodilute hydrochloric acid (0.25 M, 200 ml). The organic phase wasseparated, and washed in turn with dilute sodium hydroxide and brine,and dried (MgSO₄). The solvents was evaporated to leave a viscous brownoil, which was filtered through a pad of silica gel using ethylacetate-hexane (1:1) as eluent to give a yellowish oil, 14.3 g (98%),which solidified on standing. This material was broken up under hexaneto afford the product as a tan solid, m.p. 93-95°C. δ_(H) (400 MHz,CDCl₃) 8.22 (1H, d, J=1.5 Hz), 8.16 (1H, d, J=1.8 Hz), 7.85 (1H, s),7.73 (1H, t, J=1.2 Hz), 3.52 (3H, b s) and 3.41 (3H, bs). Analysis ofthis material by HPLC showed it to be >99.8% pure.

Preparation ofPyridin-2-yl-2,8-bis(trifluoromethyl)-4-quinolinemethanone

To a solution of the amide described above (10 g, 28.4 mmol) inanhydrous ether (100 ml) was added a solution of 2-pyridyl lithium(Pinder et al (J. Med. Chem. 1968, 11, 267)) [formed by addition of2-bromopyridine (3.3 ml, 34.6 mmol) to a solution of butyl lithium (29.7ml of a commercial 1.6 M solution, diluted with an equal quantity ofether) at −78°C.] at −78° C. Analysis of the reaction by TLC after 10min showed that no starting material remained. The reaction was allowedto warm to room temperature, then poured into aqueous ammonium acetate,and extracted with ether (2×100 ml), the combined organic layers washedwith brine and dried (MgSO₄). Filtration through a pad of silica gelusing ethyl acetate-hexane (1:1) afforded 9.0 g (84%) of the crudeproduct. This was recrystallised from isopropyl alcohol to give theproduct as colourless needles, identical to that described in theliterature (Hickmann et al; Pinder et al; Ohnmacht et al; and Adam etal. (Tetrahedron 1991, 36, 7609)).

Preparation of(R*,S*)-(±)-α-2-Piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol(Mefloquine)

This compound was prepared according to the literature procedure(Hickmann et al; Pinder et al; and Ohnmacht et al) to give an 85:15 mixof the erythro:threo isomers (HPLC). Recrystallisation of the crudematerial from acetonitrile afforded the erythro isomer as a powderywhite solid in >98% purity (HPLC).

Resolution of erythro enantiomers was performed according to the knownprocedure (Carroll, F. I. and Blackwell, J. T., J.Med.Chem., 1974, 17,210-219).

Measurement of Convulsant or Proconvulsant Effects

Several animal seizure models are available for the screening andcharacterisation of convulsant, proconvulsant and anticonvulsant drugs.Most models employ a chemical convulsant to induce seizures and theconvulsant effects of novel compounds are measured in terms of theirability to change the dose of convulsant required to induce a seizureresponse (or to alter the latency to seizure onset following a bolusdose of the convulsant) Most chemical convulsants work by blocking theneurotransmitter function of gamma-aminobutyric acid (GABA), thepredominant inhibitory neurotransmitter in the mammalian brain. This canbe achieved by blocking the postsynaptic action of GABA usingpentylenetetrazol or bicuculline, or via a presynaptic action using aGABA synthesis inhibitor such as 3-mercaptopropionic acid, an inhibitorof glutamate decarboxylase (GAD) to decrease GABA release into thesynapse.

Potentiation of Pentylenetetrazole-Induced Seizures to AssessPro-convulsant Effects Produced by a Test Compound in Mice

Seizure thresholds were determined in unrestrained animals following theinsertion of an infusion cannula into a lateral tail vein. The infusioncannulas comprised lengths of polythene tubing (Portex 800/100/100/100for mice fitted with hypodermic needles removed from their plastic Luerholders (Gillette 25G or 23G respectively).

Once inserted into the tail vein these cannulas did not require anyadditional attachment. The incidence of cannulas becoming dislodgedduring the seizures was extremely low (approximately 2% of the trials).An infusion pump was used to deliver the convulsant drug(Pentylenetetrazole, 12 mg/ml) dissolved in isotonic saline at aconstant rate 0.15 ml/min (mice). The animals were placed in atransparent Perspex enclosure for observation and the times to onset ofclonic and tonic seizures were measured. The criteria used to definethese events were: myoclonus accompanied by abrupt loss of rightingreflex (clonic) and rigid extension of the fore-limbs (tonic).

Measurement of Overt Physiological and Behavioral Effects in Mice Usingthe Irwin Screen

Animals were weighed, coded and placed in cages, and then allowed tohabituate to the environment for 20 minutes. Five animals were allocatedper treatment group. Body temperature was recorded (rectally in themouse) prior to drug administration. The compound was administered atvarious doses, including a vehicle control group. Treatment groups werecoded so that the operator was unaware of drug or dose given.

Animals were placed in Perspex chambers for observations at 20, 60, 180minutes after injection for a period of 10-15 minutes. If the compoundwas administered intravenously, animals were placed into Perspexchambers immediately after injection for observation. Body temperatureand any behavioral changes were recorded.

Pilot studies were carried out initially to determine doses of compoundto be used in an experiment. Only 3 animals were used per dose group. Aninitial low dose was administered and subsequent doses given were higheror lower depending on effects produced. Time of onset of drug action andmaximum effects were also recorded.

Observations made were categorised in qualitative terms. A checklist ofthe behavioral and physiological signs used to assess overt effects oftest compounds is shown in Table 1.

TABLE 1 Check-List of Observations for Irwin Screening AUTONOMIC OVERTBEHAVIOUR OBSERVATIONS increased activity piloerection decreasedactivity diarrhoea sedation exophthalmos tremor salivation convulsionsvasoconstriction  -myoclonic vasodilatation  -tonic/clonic cyanosisstraub tail lacrymation stereotypy ptosis increased exploration miosisdecreased exploration mydriasis ataxia weakness hypothermia catalepsypain threshold (tail pinch) loss of righting reflex writhing (i.p.adminstration)

Biological Results

The behavioral effects of the two enantiomers of mefloquine wereassessed following daily dosing (30,100,300 mg/kg p.o) in mice using anIrwin Screen. Clear qualitative differences were observed between thetwo enantiomers, with the (−)-enantiomer appearing more potent ineliciting spontaneous seizures, irritability and aggressiveness.Twenty-four hours after the first dose, animals given 300 mg/kg of the(−)-enantiomer were difficult to handle and displayed aggression towardcage-mates. After 48 hr, the same group of animals displayed spontaneousclonic seizures, increased reactivity, and reduced body weight. Withinfive days, the high dose (−)-enantiomer treated animals showed reducedactivity, unkempt appearance and assumed a hunched body posture.Although the latter effects were observed in animals treated with the(+)-enantiomer, these effects only became apparent after 5-6 days, anddid not include seizures.

In a subsequent study to assess the possibility of a proconvulsanteffect of the (−)-enantiomer, it was found that 24 hrs after a singleinjection of either the (+)- or (−)-enantiomer (30, 100, 300 mg/kg p.o),a dose of 300 mg/kg of the (−)-enantiomer significantly reduced thelatency to onset of clonic seizures induced by infusion of theconvulsant drug pentylenetetrazole (PTZ). These observations areconsistent with the reported neuropsychiatric side-effects of mefloquinein humans, and indicate that the (+)-enantiomer of mefloquine hassignificantly reduced side-effects.

Adenosine Receptor Interaction

The mefloquine (+)- and (−)-enantiomers were initially examinedseparately in an assay measuring in vitro binding to adenosine receptorsby determining the displacement of [³H]-NECA binding to bovine striatalmembranes. The data are shown in FIG. 1 and Table 2.

TABLE 2 Binding of the mefloquine (+)- and (−)- enantiomers to bovinestriatal adenosine receptors (displacement of [³H]-NECA). MECA is shownas control Ki MECA 58 nM (+)-enantiomer 38 μM (−)-enantiomer 83 nM

Once it was established that the (−)-enantiomer of mefloquine exhibiteddisplacement of [³H]-NECA in bovine striatum, the adenosine receptorsubtype binding was examined using radioligand binding assays employingthe selective P₁ radioligands [³H]-DPCPX (adenosine A₁ receptor),[³H]-CGS 21680 (adenosine A_(2A) receptor) and [¹²⁵I]AB-MECA (adenosineA₃ receptor). The results are summarised in Table 3, with comparativeliterature data for other adenosine receptor ligands. The full doseresponse curves are shown in FIGS. 2-4.

TABLE 3 Binding of mefloquine (+)- and (−)-enantiomers to adenosine A₁,A_(2A) and A₃ receptors BINDING ASSAY CONDITIONS Adenosine A₁ AdenosineA_(2A) Adenosine A₃ Source Rat Brain Human Human Ligand 0.14 nM [³H]-4.0 nM [³H]-CGS 0.4 nM [¹²⁵I]AB- DPCPX 21680 MECA Non-Specific 10 μM PIA10 μM NECA 10 μM NECA RESULTS (K_(i)) (+) enantiomer  6.4 μM  1.8 μ M  7.7 μM (−) enantiomer 202 nM  4.4 nM   6.8 μM Reference Adenosinereceptor agonists NECA  17 nM  19 nM  110 nM CHA  26 nM 510 nM 7700 nMReference Adenosine A_(2A) receptor antagonists KW-6002^(†) 580 nM  13nM na KF17837^(†) 390 nM  8 nM na ZM241385^(‡)  2.0 μM  0.3 nM  151 μM^(†)Shimada et al., BioMed. Chem. Lett., 1997, 7, 2349-2352. ^(‡)Poucheret al., Br. J. Pharm., 1995, 115: 1096-1102

The above data lead to the conclusion that the mefloquine (−)-enantiomeris a selective ligand for the adenosine A_(2A) receptor subtype invitro. The binding of the (+)-enantiomer is over two orders of magnitudeweaker.

Assessment of the Interaction of the (+)-Enantiomer and the(−)-Enantiomer With NECA in PC-12 Cells

PC-12 cells (purchased from ECACC) were maintained in RPMI 1640 media(Sigma) supplemented with 2 mM L-glutamine and 10% FCS in a controlledenvironment of 5% CO₂, 95% humidity at 37° C.

On the day of the assay the cells were gently harvested and centrifugedat 700 RPM for 10 min. The supernatant was discarded and the pelletre-suspended to a final density of approx. 1×10⁶ cells/ml in modifiedRPMI 1640 media. 600 μl of this suspension was mixed with 200 μl ofmolten agarose cell entrapment media (Molecular Devices) pre-incubatedto 37° C. The cell suspension was returned to the incubator for afurther 10 minutes for equilibration. 10 μl of this cell agarosesuspension was spotted onto the middle of the capsule insert well andallowed to set before being placed in the sensor chambers in thecytosensor® microphysiometer. The cells were maintained on theinstrument with the low buffering capacity modified RPMI 1640 media(Molecular Devices) and their base line extracellular acidificationrates allowed to settle.

Agonist concentration curves were constructed by exposing the cells toincreasing concentrations of agonist for a total exposure time ofapproximately 1 min 53 sec. Agonist concentration curves in the presenceof antagonists were performed following pre-incubation of the cells tothe antagonists for at least 20 min prior to agonist exposure (in thepresence of the fixed antagonist concentration) Baseline acidificationrates were normalised to 100%, and all responses were calculated as a %increase over normalised baseline.

All data was fitted to Log-concentration response curves to calculatehalf maximally effective concentrations (EC₅₀ values) by non-linearregression using the Graph Pad Prism software package.

The results (shown in FIG. 5) indicate that the (−)-enantiomer,(−)-(11S,2′R)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol,binds to adenosine receptors as an antagonist. PC12 cells endogenouslyexpress adenosine A_(2A) receptors and have been used as a source offunctionally-coupled A_(2A) receptors. FIG. 6 shows that similar resultsare obtained when CGS 21680, a selective A_(2A) receptor agonist, isused instead of NECA. The antagonist effect of the (−)-enantiomer isspecific to adenosine A_(2A) receptors.

What is claimed is:
 1. A method of treating or preventing malaria withreduced side-effects compared to the racemic(±)-(R*,S*)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol,comprising administering to a subject in need of such treatment aneffective dose of(+)-(11R,2′S)-α-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanolor a pharmaceutically acceptable salt thereof substantially free of its(−)-enantiomer.
 2. A method according to claim 1, wherein said subjectis human.
 3. A method according to claim 1, which further comprisesadministering one or more other anti-malarial drugs to said subject. 4.A method according to claim 3, wherein said anti-malarial drugs areselected from the group consisting of chloroquine, pyrimethamine,sulfadoxine, amodiaquine, quinine/quinidine, halofantrine,artemether/artesunate, tovaquone, proguanil, doxycycline and dapsone. 5.A method according to claim 1, wherein the subject is undergoing therapywith a chemical compound which interacts with purinergic receptors.
 6. Amethod according to claim 5, wherein the subject is undergoing therapywith a drug selected from the group consisting of theobromine,theophylline, carbamazepine, papaverine or dipyridamole.
 7. A methodaccording to claim 5, wherein the subject consumes ethanol.
 8. A methodaccording to claim 2, wherein the subject has, or is at risk from, amedical condition related to a disorder of purinergic receptor function.9. A method according to claim 8, wherein the medical condition is aneuropsychiatric disorder.
 10. A method according to claim 9, whereinthe medical condition is depression.
 11. A method according to claim 9,wherein the medical condition is an anxiety disorder.
 12. A methodaccording to claim 11, wherein the medical condition is a panicdisorder.
 13. A method according to claim 9, wherein the medicalcondition is a sleep disorder.
 14. A method according to claim 8,wherein the medical condition is a seizure disorder.
 15. A methodaccording to claim 8, wherein the medical condition is a cardiovasculardisorder.
 16. A method according to claim 15, wherein the medicalcondition is a diagnosed cardiovascular disorder.
 17. A method accordingto claim 15, wherein the medical condition is ischaemic heart disease.18. A method according to claim 15, wherein the medical condition iscongestive heart failure.
 19. A method according to claim 15, whereinthe medical condition is cerebrovascular disease.
 20. A method accordingto claim 19, wherein the medical condition is transient ischaemicattacks.
 21. A method according to claim 16, wherein the subject isundergoing treatment with beta-blocker drugs.
 22. A method according toclaim 16, wherein the subject is undergoing treatment with avasodilator.
 23. A method according to claim 16, wherein the medicalcondition is arrhythmia or a related disorder.
 24. A method according toclaim 16, wherein the subject is undergoing treatment with nifedipine.25. A method according to claim 8, wherein the medical condition is adigestive disorder.
 26. A method according to claim 25, wherein thesubject is undergoing treatment with a proton pump inhibitor.
 27. Amethod according to claim 8, wherein the medical condition is abronchial disorder.
 28. A method according to claim 27, wherein themedical condition is asthma.
 29. A method according to claim 28, whereinthe subject is undergoing treatment with theophylline.
 30. A methodaccording to claim 27, wherein the medical condition is chronicobstructive pulmonary disease.
 31. A method according to claim 8,wherein the medical condition is erectile dysfunction.